Mouse model of severe recessive RYR1-related myopathy

Abstract

Ryanodine receptor type I (RYR1)-related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype of childhood onset non-dystrophic muscle disorders. There are no treatments for this severe group of diseases. A major barrier to therapy development is the lack of an animal model that mirrors the clinical severity of pediatric cases of the disease. To address this, we used CRISPR/Cas9 gene editing to generate a novel recessive mouse model of RYR1 RM. This mouse (Ryr1TM/Indel) possesses a patient-relevant point mutation (T4706M) engineered into 1 allele and a 16 base pair frameshift deletion engineered into the second allele. Ryr1TM/Indel mice exhibit an overt phenotype beginning at 14 days of age that consists of reduced body/muscle mass and myofibre hypotrophy. Ryr1TM/Indel mice become progressively inactive from that point onward and die at a median age of 42 days. Histopathological assessment shows myofibre hypotrophy, increased central nuclei and decreased triad number but no clear evidence of metabolic cores. Biochemical analysis reveals a marked decrease in RYR1 protein levels (20% of normal) as compared to only a 50% decrease in transcript. Functional studies at end stage show significantly reduced electrically evoked Ca2+ release and force production. In summary, Ryr1TM/Indel mice exhibit a post-natal lethal recessive form of RYR1 RM that pheno-copies the severe congenital clinical presentation seen in a subgroup of RYR1 RM children. Thus, Ryr1TM/Indel mice represent a powerful model for both establishing the pathomechanisms of recessive RYR1 RM and pre-clinical testing of therapies for efficacy.

Figure 1

Generation of the Ryr1^TM/Indel mouse line. (A) Using CRISPR/Cas9, a threonine-to-methionine missense mutation (T4709M) and a 16 bp deletion (Indel) were generated in exon 96 of the mouse Ryr1 locus. ‘y’ indicates a heterozygous base change from C to T. (B) Representative Sanger sequencing results showing the T4709M substitution and 16 bp deletion mutations. (C) Breeding strategy of crossing heterozygous Ryr1^WT/TM mice with Ryr1^WT/Indel mice to obtain compound Ryr1^TM/Indel mice.

Figure 2

Ryr1^TM/Indel mice exhibit reduced body weight and premature death. (A) Ryr1^TM/Indel mice (40 days old) exhibit reduced size. Ryr1^TM/Indel mice also show hunched posture and splayed hindlimbs compared to Ryr1^WT/WT mice. Depicted is a photograph of representative Ryr1^WT/WT and Ryr1^TM/Indel littermate mice at 40 days. (B) Reduced body weight of Ryr1^TM/Indel mice, noticeable at 14 days postnatal, progresses with age (n = 8–9). (C) Ryr1^TM/Indel mice exhibit decreased survival. Kaplan–Meier survival analysis reveals that Ryr1^TM/Indel mice do not survive longer than 57 days with a median survival of 42 days (n = 25 for each group). (D) X-ray analysis reveals both scoliosis (lateral curve, top panel) and spinal kyphosis (arrow, bottom panels) in Ryr1^TM/Indel mice compared to Ryr1^WT/WT mice.

Figure 3

Ryr1^TM/Indel mice exhibit in vivo muscle weakness assessed by wire hang test. (A) Average combined forelimb and hindlimb grip strength (normalized to body weight) in 55-day-old Ryr1^WT/WT (circles) and Ryr1^TM/Indel mice (squares). No statistically significant difference was observed; Ryr1^TM/Indel exhibit an overall grip strength of 8.40 ± 0.25 (N/g) (n = 5, 3 male and 2 female) and Ryr1^WT/WT an average of 9.14 ± 0.25 (N/g) (n = 4, 3 male and 2 female). (B) Wire hang test of 55-day-old Ryr1^WT/WT (circles) and Ryr1^TM/Indel mice (squares). Ryr1^TM/Indel exhibit significantly decreased average [±standard error of the mean (SEM)] wire hang time in spite of a lower body weight (average = 36.4 ± 16.1 s, n = 6) compared to Ryr1^WT/WT mice (average = 122.5 ± 17.1 s, n = 7) (^**P = 0.0039).

Figure 4

Reduced twitch and tetanic-specific force production in EDL muscles from Ryr1^TM/Indel mice. (A, B) Representative superimposed specific force traces elicited during twitch (A) and tetanic (B) stimulation. (C, D) Average (±SEM) peak twitch (C) and tetanic-specific (D) force for each genotype. (E, F) Analysis of specific (E) and relative (F) force–frequency curves from the same muscles shown in (A)–(D). Number of muscles analysed: Ryr1^WT/WT = 12 (6 mice); Ryr1^WT/indel = 16 (8 mice); Ryr1^WT/TM = 6 (3 mice); Ryr1^TM/indel = 8 (4 mice).

Figure 5

Histological analysis of Ryr1^TM/Indel mice reveals reduced myofibre size and increased percent of central nuclei. Histopathologic analysis of cross sections for TA muscle of 55-day-old Ryr1^WT/WT and Ryr1^TM/Indel mice. (A) As seen with H/E stain, fibres from Ryr1^TM/Indel mice compared to Ryr1^WT/WT mice exhibit decreased fibre size and reveal the presence of central nuclei. Scale bar is 50 μm. (B) As seen with SDH stain, muscles from Ryr1^TM/Indel mice do not exhibit cores or core-like lesions. Scale bar is 50 μm. (C) Mean (±S.E.M.) myofibre diameter of Ryr1^TM/Indel (n = 4, male) and Ryr1^WT/WT mice (n = 6, 4 male and 2 female). Average fibre diameter is 42.9 ± 2.6 μm versus 32.2 ± 1.1 μm in Ryr1^WT/WT and Ryr1^TM/Indel mice, respectively (^*P = 0.0189). (D) Percent of myofibres containing central nuclei in Ryr1^TM/Indel (n = 5) and Ryr1^WT/WT mice (n = 5). Average (±SEM) percent central nuclei is 0.00% and 0.78% ± 0.03% in Ryr1^WT/WT and Ryr1^TM/Indel mice, respectively (^****P < 0.0001).

Figure 6

Ryr1 transcript levels are reduced in quadriceps muscle of Ryr1^WT/Indel and Ryr1^TM/Indel mice. (A) Representative agarose gel of Ryr1 (lower band) and Gapdh loading control (upper band) amplification products. (B, C) Quantitative analysis of Ryr1 (B) and DHPR (C) transcript levels in quadriceps muscles obtained by semiquantitative PCR using 6-FAM-labelled primers. Data are shown as mean ± SEM from five Ryr1^WT/WT, six Ryr1^WT/Indel, three RyR1^WT/TM and three Ryr1^TM/Indel mice (^*P < 0.05).

Figure 7

RyR1 protein level is markedly reduced in TA muscle from Ryr1^TM/Indel mice. (A) Representative western blots gels for RyR1, DHPR, SERCA and GAPDH in TA muscle from each genotype. Loaded per lane was 5 μg of total TA lysate. (B) Quantitation of relative RyR1 level (normalized to GAPDH) for each genotype. (C) Quantitation of relative DHPR level (normalized to GAPDH) for each genotype. (D) Quantitation of relative SERCA level (normalized to GAPDH) for each genotype. Data are shown as mean ± SEM from seven Ryr1^WT/WT, eight Ryr1^WTInde, three Ryr1^WT/TM and four Ryr1^TM/Indel mice (^*P < 0.001).

Figure 8

Reduced twitch and tetanic Ca²⁺ release in FDB fibres from Ryr1^TM/Indel mice. (A, B) Representative superimposed changes in relative mag-fluo-4 fluorescence during electrically evoked twitch (A) and tetanic (B) stimulation. (C, D) Average (±SEM) peak twitch (C) and tetanic (D) change in relative mag-fluo-4 fluorescence for each genotype compared to Ryr1^WT/WT. Data are shown as mean ± SEM in fibres from Ryr1^WT/WT (n = 92), Ryr1^WT/Indel (n = 107), RyR1^WT/TM (n = 43) and Ryr1^TM/Indel (n = 60) mice (^*P < 0.001).

Figure 9

Ryr1^TM/TM mice exhibit a hyperthermic response during isoflurane exposure. (A) Representative time course of core temperature changes in adult Ryr1^WT/WT, Ryr1^WT/TM, Ryr1^TM/TM and Ryr1^WT/YS mice during 25 min exposure to isoflurane. (B) Average (±SEM) maximal change in core temperature during isoflurane exposure for each of the genotypes tested. Data are shown as mean ± SEM from 4 Ryr1^WT/WT, 11 Ryr1^WT/TM, 3 Ryr1^TM/TM and 3 Ryr1^WT/YS mice (^*P < 0.05 compared to Ryr1^WT/WT).